The F-actin-depolymerizing factor (ADF) of human serum was purified and identified as a 93000-daltons protein. The concentration of ADF was calculated to 120-150 micrograms/ml in four normal sera and about half as much in three sera from leukemia patients treated with cytostatic drugs. In the presence of Ca2+ ADF shortened actin filaments into fragments, the size of which was correlated to the actin: ADF molar ratio, as judged by electron microscopy. The severing of F-actin was not necessarily followed by an increase in the quantities of monomeric actin, as determined by a DNase I inhibition assay and a sedimentation assay. The findings indicated that ADF shortens filamentous actin by breaking bonds between adjacent actin molecules thereby forming stable ADF-actin complexes, without a monomeric net release. The effect of ADF on F-actin was rapid and was reversed upon chelation of Ca2+. ADF cross-reacted immunologically and exhibited similarity in reaction mechanism with gelsolin, the Ca2+-dependent F-actin-severing protein from macrophages. This implies that the proteins are both structurally and functionally related. The physiological role of ADF may be to handle actin released at cell destruction, probably by forming ADF-G-actin 1:1 complexes thereby preventing formation of actin filaments.